agglutination methods Flashcards by Sharmaine Madjid

visible expression of the aggregation of antigens and antibodies

Precipitation and agglutination

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aggregation of soluble test antigens

Precipitation

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soluble antigen + soluble antibody =

visible insoluble complex

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whereby specific antigens (e.g., red blood cells) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum

Agglutination

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In Agglutination…

_______ may be needed to indicate visibly that an antigen-antibody reaction has taken place (latex particles and colloidal charcoal)

Artificial carrier particles

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easy to perform; most sensitive tests currently available

Agglutination

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first agglutination tests were developed

1960s

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types of agglutination

Latex Agglutination
Flocculation Tests
Direct Bacterial Agglutination
Hemagglutination

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(1)

bound to the surface of latex beads (C-reactive protein)
forming visible cross-linked aggregates of latex beads and antigen

Antibody molecules

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(1)

coated the latex particles (pregnancy testing, rubella antibody testing)
agglutinate into large visible clumps

Antigen

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Variations of latex agglutination

Coagglutination
Liposome-enhanced Testing

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(1)

Uses antibodies bound to a particle to enhance the visibility of agglutination
Highly specific method
Not as sensitive as latex agglutination

Coagglutination

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detect minute amounts of human chorionic gonadotropin (hCG)

LATEX AGGLUTINATION: PREGNANCY TESTING

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a glycoprotein hormone secreted by the trophoblast of the developing embryo

hCG

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first 6 to 8 weeks after conception: helps maintain the ______ and stimulate the production of ______

corpus luteum ; progesterone

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hCG two noncovalently linked subunits

a unit and B subunit

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identical to luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH)

a-unit

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many pregnancy test kits contain ________ directed against the ______ to increase the _______ of the reaction

monoclonal antibody (MAb) ; β subunit ; specificity

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level of hCG should double every ___ to ___ days

2 to 3

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incubation of the patient’s specimen with anti-hCG, then addition of latex particles coated with hCG

Agglutination Inhibition

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agglutination inhibition reaction results

(+) rxn: no agglutination (neutralizes the antibody)
(-) rxn: agglutination

If hCG is present, it neutralizes the antibody; thus,
no agglutination of latex particles is seen. If no
hCG is present, agglutination occurs between the
anti-hCG and hCG-coated latex particles.

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latex agglutination that is rapid, direct, uses monoclonal Antibodies

Pregnancy Latex Slide Agglutination

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Pregnancy Latex Slide Agglutination reaction results

(+) rxn: agglutination within 2 mins
(-) rxn: no agglutination

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In pregnancy latex agglutination..

agglutination should be read within _______ to avoid erroneous results caused by _______

3 minutes ; evaporation

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False-Positive Results (latex agglutination pregnancy testing)

- hCG injection (Pregnyl): can remain in the patient’s system for as long as 10 days - Chorioepithelioma - hydatidiform mole - excessive ingestion of aspirin - testicular tumor

False-Negative Results (latex agglutination pregnancy testing)

Testing before reaching detectable levels of hCG

(2) for antibody detection

FLOCCULATION TESTS

(2) soluble antigen + antibody =

formation of a precipitate of fine particles

used in syphilis serologic testing

FLOCCULATION TESTS

tests in the flocculation tests..

VDRL (Venereal Disease Research Laboratories) Test RPR (Rapid Plasma Reagin) Test

- antibody-like protein, reagin, binds to the test antigen, cardiolipin-lecithin–coated cholesterol particles, - produces the particles that flocculate

Venereal Disease Research Laboratories (VDRL) test

antigen, cardiolipin-lecithin–coated cholesterol with choline chloride, also contains charcoal particles that allow for macroscopically visible flocculation

Rapid Plasma Reagin (RPR) Test

Used to detect antibodies directed against the pathogens, test produced by the host to antigen determination on the surface of bacterial agent in response to infection within that bacterium.

Direct Bacterial Agglutination

(3) In a thick suspension of the bacteria, the binding of specific antibodies to surface antigens of the bacteria causes the bacteria to clump together in visible aggregates. This type of agglutination is called

Bacterial Agglutination

use of _______ enhances the aggregation of bacteria since most bacterial surfaces exhibit a _______ that causes them to repel each other

sterile physiologic saline ; negative charge

_______ is considered more sensitive than slide testing because it allows more time for the antigen-antibody reaction

tube testing

(4) detects antibodies to erythrocyte antigens

HEMAGGLUTINATION

HEMAGGLUTINATION specimen

contains Ab

- detect antibodies to antigens other than those present on the cells - used to cross-link antigens to the cells: chromic chloride, tannic acid, and glutaraldehyde

Indirect hemagglutination or passive hemagglutination (PHA)

______ is the clumping of particles that have antigens on their surface

Agglutination

Mechanisms of Agglutination

Sensitization Lattice Formation

- First phase - physical attachment of antibody molecules to antigens on the erythrocyte membrane - combination of antigen and antibody is a reversible chemical reaction - Elution: when physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium

Sensitization

In sensitization, combination of antigen and antibody is a _______

reversible chemical reaction

when physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium

Elution

Inert particles such as latex, RBCs, and bacteria have a NET NEGATIVE SURFACE CHARGE called the ________

zeta potential

a technique to reduce zeta potential that removes negatively charged sialic acid residues from cell surface membrane

Enzyme pretreatment of red blood cells

a technique to reduce zeta potential that increases electrical conductivity of environment

Addition of colloids (e.g., albumin)

a technique to reduce zeta potential which action is mechanical process to force red blood cells closer together.

Centrifugation

each antibody molecule must have at least 2 binding sites and that an antigen must be multivalent

Lattice hypothesis

Zone of equivalence=

detectable reaction

overcome by serially diluting the antibody-containing serum until optimum amounts of antigen and antibody are present in the test system

prozone phenomenon

small aggregates (clumps) are surrounded by excess antigen

postzone phenomenon

Repeat blood specimen should be collected _______ weeks later

1 or more

______ and _______ of antigenic determinants both affect agglutination

placement and number

If dissimilar antibodies with the same binding constant are directed against antigenic determinants located close to each other, antibodies will compete for space in reaching their specific receptor sites

Steric Hindrance

effect of antibody competition

antibody blocking

(4) pH of the medium used for testing:

optimum pH of 6.5 to 7.5

cold-reacting (thermal range, 4° C to 22° C)

IgM antibodies

warm-reacting, with an optimum temperature of reaction at 37° C

IgG antibodies

establishment of cross-links between sensitized particles RBCs) and antibodies, resulting in aggregation which is a slower process than the sensitization phase

Lattice Formation

Methods of enhancing agglutination

1. Centrifugation 2. Treatment with proteolytic enzymes 3. Use of colloids 4. AHG Testing

method of enhancing agglutination.. - physically forces the cells together - overcome the problem of distance by subjecting sensitized cells to a high gravitational force

Centrifugation

method of enhancing agglutination.. alters the zeta potential or dielectric constant

Treatment with proteolytic enzymes

method of enhancing agglutination.. antigens may be so deeply embedded in the membrane surface

AHG testing

Observation of agglutination: gently shaking the test tube containing the serum and cells and viewing the lower portion, the button

Graded Agglutination Reactions

Because agglutination is a reversible reaction, the test tube must be treated delicately, and _______

hard shaking

False appearance of clumping

Pseudoagglutination

Pseudoagglutination rarely occur because of _____

Rouleaux Formation

Rouleaux formation can be encountered in patients with .....

high or abnormal types of globulins in their blood (multiple myeloma or after receiving dextran as a plasma expander)

To disperse the pseudoagglutination, a few drops of ______ can be added to the reaction tube, remixed, and reexamined. (PS: performed after pseudoagglutination is suspected)

physiologic NaCl (saline)

grading agglutination reactions... D: no aggregates

Negative

grading agglutination reactions... D: A few isolated aggregates; mostly free-floating cells; supernatant appears red

Mixed field

grading agglutination reactions... D: Tiny aggregates barely visible macroscopically; many free erythrocytes; turbid and reddish supernatant.

Weak (±)

grading agglutination reactions... D: A few small aggregates just visible macroscopically; many free erythrocytes turbid and reddish supernatant

grading agglutination reactions... D: Medium-sized aggregates; some free erythrocytes; clear supernatant.

grading agglutination reactions... D: Several large aggregates; some free erythrocytes; clear supernatant.

grading agglutination reactions... D: All erythrocytes are combined into one one solid aggregate; clear supernatant.

In Microplate Agglutination Reactions, _____ and _____ allows for the performance of a large number of tests on a single plate

hemagglutination and solid-phase adherence assays

a compact plate of rigid or flexible plastic with multiple wells

microplate

- ______ well has been used most often in immunohematology - volume capacity of each well: ______

- U-shaped well - approx 0.2 mL

In microplate agglutination reactions.. Samples and reagents are dispensed with ________ pipettes

small-bore Pasteur pipettes

In microplate agglutination reactions.. After the specimens and reagents are added to the wells, they are mixed by _______ of the plates. The microplate is then _______ for an immediate ______

gentle agitation ; centrifuged ; reading

After centrifugation, the cell buttons are resuspended by _________ or by using a __________ which is a shaker provides a more consistent and standard resuspension of the cells.

gently tapping the microplate ; flat-topped mechanical shaker

Microplate Agglutination Reactions Results

(+) rxn: particles settle in a diffuse uneven button (-) rxn: smooth compact button

Reading agglutination.. Cells: No Agglutinates Supernate: Dark, Turbid, Homogenous

Grade: 0

Reading agglutination.. Cells: Many tiny agglutinates, Many free cells, May not be visible without microscope. Supernate: Dark, Turbid

Grade: w+

Reading agglutination.. Cells: Many small agglutinates, Many free cells Supernate: Turbid

Grade: 1+

Reading agglutination.. Cells: Many medium-sized agglutinates, Moderate number of free cells. Supernate: Clear

Grade: 2+

Reading agglutination.. Cells: Several large agglutinates, Few free cells Supernate: Clear

Grade: 3+

Reading agglutination.. Cells: One large, solid agglutinate and No free cells Supernate: Clear

Grade: 4+

In immunofixation electrophoresis.. ______________: exhibit faintly staining bands

Hypogammaglobulinemia

In immunofixation electrophoresis.. _________: show darkly staining bands in the gamma region

polyclonal hypergammaglobulinemia

In immunofixation electrophoresis.. __________: dark and narrow bands in specific lanes

presence of monoclonal antibody

In direct bacterial agglutination.. __________: rapid screening test useful in diagnosing typhoid fever

Widal test

employs particles that are coated with antigens not normally found on their surfaces

Passive, or Indirect, agglutination

the particles used in Passive, or Indirect, agglutination

Erythrocytes, Latex and Gelatin

found that IgG was naturally adsorbed to the surface of polystyrene latex particles.

(1955) Singer and Plotz

In Passive, or Indirect, agglutination.. ______ is attached to the carrier particle

Antigen

have been used to detect rheumatoid factor, antibodies to Group A Streptococcus antigens, and antibodies to viruses such as rotavirus, cytomegalovirus, rubella, and varicella-zoster

Latex agglutination tests

- antibody is attached to a carrier particle - often used to detect microbial antigens

Reverse Passive Agglutination

based on competition between particulate and soluble antigens for limited antibody combining sites

Agglutination inhibition

(+) R in agglutination inhibition

no agglutination

agglutination inhibition, involves _____ complexed to proteins; the _________ is then attached to a carrier particle

haptens ; hapten–protein conjugate

use the same principle, except RBCs are the indicator particles

Hemagglutination inhibition

involves measuring the number of residual nonagglutinating particles in a specimen

particle-counting immunoassay (PACIA)

These particles are counted by means of a ______ in an optical particle counter similar to the one that is designed to count blood cells.

laser beam

Measurements are made by looking at the rate at which the number of unagglutinated particles decrease, called a ______ or the total number of unagglutinated particles left at the end, known as an ________

rate assay ; end-point assay

An _________ exists between the number of unagglutinated particles counted and the amount of unknown antigen in the patient specimen

inverse relationship

physical preservation of serum

refrigerate for 72 hours at 4 to 6 degree celsius

chemical preservation of serum

add 0.001g merthiolate powder per mL serum or 5% phenol or tricresol at 0.1 mL per mL serum

physical inactivation of serum to remove complement

heat serum at 56 degree celsius for 30 mins

physical inactivation of serum for VDRL

heat serum at. 60 to 62 degree celsius for 3 to 4 mins

chemical inactivation of serum

choline chloride

soluble ag/ small ag

Ag = antigen

measurement of precipitation by light scattering = at fluid

Turbidimetry Nephelometry

measure turbidimetry/cloudiness

turbidimetry with the use of spectrophotometer

measure light scattered in a particular angle

nephelometry with the use of nephelometer

- no current used - uses agar/agarose because no negative charge

precipitation by passive immunodiffusion at solid/semi-solid medium

precipitation by passive immunodiffusion at solid/semi-solid medium advantage

cheap, simple

precipitation by passive immunodiffusion at solid/semi-solid medium disadvantage

longer turn around time (TAT)

- single diffusion, single dimension - the antibody you incorporated into agarose at test tubes - semi-quantitative - end: presence of precipitin line - thicker line = increased concentration

Ouidin Test

- Single Diffusion, Double Dimension - Uses plate with antibody - Quantitative: diameter is directly proportional to concentration - (+) R = precipitin ring

Radial Immunodiffusion (RID)

2 methods in RID

Fahey and McKelvey Method (Kinetic Diffusion) Mancini Method (Endpoint Diffusion)

- Measure diameter before the completion of the reaction. - Diameter is proportional to log of concentration.

Fahey and McKelvey Method (Kinetic Diffusion)

- measures diameter after the completion of the reaction - square of diameter is proportional to concentration - IgG: 24 hours - IgM: 50 to 72 hours

Mancini Method (endpoint diffusion)

- Qualitative ; 1st generation test for HBsAg - both Ag and Ab is moving

Ouchterlony Technique (Double diffusion, Double dimension)

Ouchterlony Technique (Double diffusion, Double dimension) pattern

Identity = smooth curve Partial Identity = spur formation Non-identity = cross/intersect

____ separates molecules using electric current

Electrophoresis

Precipitation by Electrophoretic Techniques

1. Rocket Electrophoresis (Laurell Technique) 2. Immunoelectrophoresis (IEP) 3. Immunofixation Electrophoresis 4. Counter Immunoelectrophoresis

- one dimension electroimmunodiffusion - RID + electrophoresis - total distance of antigen migration and precipitate directly proportional to antigen concentration. - (+)R: resembles rocket - increased peak = increased concentration

Rocket Electrophoresis (Laurell Technique)

Rocket Electrophoresis (Laurell Technique) advantage and disadvantage

adv: decreased TAT dadv: expensive, complex

- described by Grabar and Williams - useful for ID of monoclonal protein (Bence Jones) - double diffusion + electrophoresis

Immunoelectrophoresis (IEP)

1st diffusion: separate component trough + antisera 2nd diffusion: towards the trough

Immunoelectrophoresis (IEP)..

Immunoelectrophoresis (IEP) disadvantage

interpretation of the test result is complex

Immunoelectrophoresis (IEP) (+)R

ppt toward the trough

canal-like structure

trough

- replaced IEP - to identify and characterize serum and urine CHON - antisera is directly added to the medium - described by Alper and Johnson - ex. western blot

Immunofixation electrophoresis

ag and ab = placed on the well directly opposite to each other.

Counter electrophoresis

antibody is towards the ___ antigen is towards the ___

cathode ; anode